Two approaches have been used in efforts to discover novel chemicals useful in medicine, agriculture, or basic research. In the first approach of rational design, researchers perform structural studies to determine the three-dimensional structure of a target molecule in order to design compounds which are likely to interact with that structure. In the second approach, large libraries of compounds are screened for a desired biological activity. Compounds exhibiting activity in these screening assays become lead chemical compounds. Further study of compounds with structural similarity to the lead compounds can then lead to the discovery of other compounds with optimal activity.
Although traditional screening assays have focused on the screening of naturally occurring compounds, the ability to synthesize large combinatorial libraries of compounds with diverse structures has greatly increased the number of compounds available for screening. In combinatorial chemistry, each reactant from a first group of reactants is reacted with each reactant from a second group of reactants to yield products containing all the combinations possible from the reaction. If desired, all of the products from the first reaction are then reacted with each reactant from a third group of reactants to yield a large array of products. Additional reactions, if desired, can further increase the size of the library of compounds. Where it is desirable to use protection/deprotection protocols to prevent reactive groups from participating in a given reaction step, typically the same protocols are used for each compound in the growing library.
The generation and use of combinatorial chemical libraries for the identification of novel lead compounds or for the optimization of a promising lead candidate has emerged as a promising and potentially powerful method for the acceleration of the drug discovery process. (Terrett, N. K., et al., Tetrahedron 51:8135 (1995); Gallop, M. A., et al., J. Med. Chem. 37:1385 (1994); Janda, K. D., Proc. Natl. Acad. Sci. U.S.A. 91:10779 (1994); Pavia, M. R. et al., Bioorg. Med. Chem. Lett. 3:387 (1993)).
Combinatorial chemistry preferentially employs generally applicable reaction strategies and protocols which provide high-yielding reaction products. Various approaches to the synthesis of diverse chemical libraries have been disclosed including several methods utilizing solid supports. E.g., Bunin, B. A. & Ellman, J. A., J. Am. Chem. Soc. 114:10997-10998 (1992); Hobbes Dewitt, S., et al., Proc. Natl. Acad. Sci. USA 90:6909-6913 (1993); Chen, C., et al., J. Am. Chem. Soc. 116:2661-2662 (1994); Backes, J. B. & Ellman, J. A., J. Am. Chem. Soc. 116:11171-1172 (1994). Solid phase polymer-supported synthesis employs an insoluble matrix substrate upon which molecules in a combinatorial library may be assembled.
In solid support synthesis, a first reactant is linked to a solid support. This linkage may include a spacer linker arm connecting a functional group on the first reactant to a functional group on the solid support. Reaction of the first reactant bound to the solid support with a second reactant produces a desired product which is bound to the solid support, while unreacted second reactant remains unbound in solution.
If desired, additional reactants can be added to the product of the first reaction in subsequent reactions. The insoluble matrix substrate is washed after each elongation step. The products are then pooled and split into a second or subsequent set of parallel reaction vessels for further elongation as desired.
Some of the features of solid phase synthesis responsible for its widespread use in chemical synthesis are its repetitive coupling reactions as well as ease of product isolation and sample manipulation. Because the growing product is bound to the solid support, unreacted reactants can be easily removed by washing and/or filtration after each reaction in the synthesis of the final product. Furthermore, because of the ease of removal of unreacted reactants, the synthesis and separation of product from unreacted reactants can be automated. In addition, the ability to isolate the resin bound product by simple filtration permits the use of large reagent excesses to obtain high yields which are required for each step of a multistep synthesis.
In part, because of these features of solid phase synthesis, solution phase combinatorial synthesis has not yet gained wide acceptance as an alternative to solid phase synthesis. There have been, however, reports of solution phase, single-step amide, ester or carbamate condensations in the preparation of library mixtures. (Pirrung, M. C. and Chen, J. J. Am. Chem. Soc. 117:1240 (1995); Smith, P. W., et al., Bioorg. Med. Chem. Lett. 4:2821 (1994); Peterson, J. B. in Exploiting Molecular Diversity: Small Molecule Libraries for Drug Discovery, La Jolla, Calif., (Jan. 23-25, 1995).
Liquid phase synthesis has features which make it attractive for use in chemical synthesis. Liquid phase synthesis does not have the drawbacks associated with heterogeneous reaction conditions which can occur in solid phase synthesis. These drawbacks include nonlinear kinetic behavior, unequal distribution and/or access to the chemical reaction, solvation problems, the use of insoluble reagents or catalysts; and pure synthetic problems associated with solid phase synthesis. Liquid phase synthesis also does not have the restrictions of scale of reaction imposed by high cost and difficulty in handling large amounts of solid support necessary to obtain large quantities of product.
Liquid phase synthesis also does not require the use of specialized protocols for monitoring the individual steps of a multistep solid phase synthesis. (Egner, B. J., et al., J. Org. Chem. 60:2652 (1995); Anderson, R. C., et al., J. Org. Chem. 60:2650 (1995); Fitch, W. L., et al., J. Org. Chem. 59:7955 (1994); Look, G. C., et al., J. Org. Chem. 49:7588 (1994); Metzger, J. W., et al., Angew. Chem., Int. Ed. Engl. 32:894 (1993); Youngquist, R. S., et al., Rapid Commun. Mass Spect. 8:77 (1994); Chu, Y. H., et al., J. Am. Chem. Soc. 117:5419 (1995); Brummel, C. L., et al., Science 264:399 (1994); Stevanovic, S., et al., Bioorg. Med. Chem. Lett. 3:431 (1993)).
In solid phase synthesis, immobilized reactants which fail to react cannot be separated from immobilized reaction product intermediates. If the unreacted reactants participate in later reactions, they will give rise to a different undesired product than the intermediates, and the desired product will be released in an impure state. Thus, to be useful, each reaction in a solid phase synthesis must proceed with an unusually high efficiency. Optimization of the reactions to obtain the required reaction efficiencies is both time consuming and challenging. Even a modest level of purity in the final product (85%) pure requires a 92% yield at each step of a two-step reaction sequence, and a 95% yield at each step of a three-step reaction sequence. These high yields are not routinely available and require both an extensive investment in reaction optimization and/or a purification of the released solid phase product at each step. In addition, it may be necessary to use capping reactions at each step of the reaction to prevent the unreacted reactant from participating in subsequent reactions.
Because intermediates are not immobilized in liquid phase synthesis, liquid phase synthesis permits ease of sample manipulation, the purification of intermediates at each step, and a homogeneous reaction conditions. The non-limiting scale, expanded and nonlimiting repertoire of chemical reactions, direct production of soluble intermediates and final products for assay or for purification make solution phase combinatorial synthesis an attractive alternative to solid phase synthesis.
There is still a need, however, for a method of combinatorial synthesis which combines the advantages of both solid phase and liquid phase synthesis. Outside the field of combinatorial chemistry, biphasic oligomeric supports have been used for the serial synthesis of individual molecules.
In biphasic oligomer-supported liquid phase synthesis the growing product is attached to a large soluble polymeric group. The product from each step of the synthesis can then be separated from unreacted reactants by precipitating the oligomer attached to the growing product. Unreacted reactants can then be easily separated from the solid phase oligomer and attached product. This permits reactions to take place in homogeneous solutions, as well as eliminating tedious purification steps associated with traditional liquid phase synthesis.
Polyethylene glycol (PEG) is a conventional biphasic support employed in the area of serial chemistry due to its favorable physical and chemical properties. PEG polymers are available in a variety of molecular weights from 2,000 to 20,000 Dalton and can be purchased from commercial sources such as Fluka, Sigma and Aldrich (St. Louis, Mo.) as unprotected or protected, mono or difunctionalized polymers (e.g. the monomethyl ether of PEG).
PEG products remain soluble in most reaction mixtures and organic solvents (w/v: benzene 10%, CCl.sub.4 10%, Dioxane 10%, Methanol 20%, Pyridine 40%, CHCl.sub.3 47%, CH.sub.2 Cl.sub.2 53%, H.sub.2 O 55%, EtOH 20% 34.degree. C., EtOH 1% 32.degree. C., EtOH 0.1% 20.degree. C., diethylether 0.01%); yet, PEG can also be precipitated by exposure to diethyl ether. This permits easy separation of product from reactants, and subsequent crystallization in cold 20.degree. C. ethanol. Unlike other polymers, PEG avoids the tendency to form gelatinous precipitates.
PEG has been employed as a biphasic support in connection with the serial synthesis and purification of oligonucleotides, oligosaccharides and peptides. The PEG is generally connected to the core molecule through an ester linkage (e.g. a free hydroxyl on PEG is esterified via a succinate linkage to a free hydroxyl on the core molecule). Amide linkages and ether linkages have also been used to link the PEG to the core molecule.
Methods for carrying out liquid phase synthesis of libraries of peptides and oligonucleotides coupled to a biphasic oligomeric support have been described. (Bayer, Ernst, et al., Peptides: Chemistry, Structure, Biology, 426-432; Bayer, Ernst & Mutter, Manfred, Nature 237:512-513 (1972); Bayer, Ernst, et al., J. Am. Chem. Soc. 96:7333-7336 (1974); Bonora, G. M. et al., Nucleosides and Nucleotides 10:269 (1991); Bonora, G. M., et al., Nucleic Acids Res. 8:3155-3159 (1990)).
Bayer & Mutter, supra, demonstrated the use of PEG as a biphasic support with respect to the synthesis of peptides. (Bayer et al., Nature 237:512 (1972).) Bayer & Mutter also discovered that the solubilizing power of PEG was sufficient to enable the synthesis of oligomers with chain lengths up to 12 residues. In addition, Bayer & Mutter noted that for short peptides of 10-15 amino acid residues, the physio-chemical properties of the PEG-bound peptides were governed not by the nature of the attached peptide, but by the nature of the polymeric ester group used as a linker (e.g., succinate linkage). PEG exhibits high retention of its crystalline phase after the attachment of short peptide blocks, but the physio-chemical properties of PEG-bound peptides for longer peptides (having more than 20 residues) strongly depended upon the primary sequence, side-chain protection and conformation of the attached peptide.
Bonora et. al., supra, carried out a large scale dideoxynucleotide synthesis using PEG as a biphasic support and obtained high yields above 90% for the synthesis of a octanucleotide with the sequence: d(TAGCGCTA). Nucleosides and nucleotides 10:269 ( 1991). Bonora also used a PEG biphasic support to synthesize cyclic oligodeoxyribonucleotides. (Bonora et al. Nucleosides and Nucleotides 12:21 (1993).)
Krepinsky et al. prepared milligram quantities of small PEG linked disccharides, utilizing the crystallization purification properties of PEG. (Krepinsky et al., J. Am. Chem. Soc. 113:5095 (1991).) Krepinsky et al. demonstrated that when the PEG was bound to a carbohydrate hydroxyl, the glycosylation reaction could be driven to virtual completion by repeated additions of the glycosylating agent. The excess reagents were subsequently washed off the precipitated PEG-bound product and the process was repeated until the desired length polymer was obtained. (Krepinsky et al., J. Am. Chem. Soc. 113:5095, 1991.)
PEG (polyethylene glycol) is a preferred biphasic support for serial syntheses due to its ease of precipitation and crystallization properties. However, alternative biphasic supports are also known in the serial chemistry area. Alternative biphasic supports include polyvinyl alcohol and polyvinylamine copolymerized with polyvinyl-pyrrolidone, etc. (Bayer et al., Nature 237:512 (1972).)
New methods of combinatorial chemistry which combine the advantages of liquid and solid phase synthesis will aid in drug discovery efforts.
Two biologically important peptides have been the focus of drug discovery efforts. The first peptide consists of a three amino acid sequence, arginine-glycine-aspartic acid. This peptide is also denoted by the single-letter amino acid code RGD. The RGD peptide is involved in cell attachment activities which play a role in diseases and conditions such as cardiovascular disease, cancer, osteoporosis, and inflammation. The second peptide consists of the sequence glutamic acid-leucine-arginine, denoted ELR. The ELR peptide is involved in conditions resulting from inflammatory responses such as rheumatoid arthritis, asthma, and acute respiratory distress syndrome (ARDS).
Interest in the RGD peptide has grown as the result of studies of the large glycoproteins from the extracellular matrix (ECM). The cell attachment activity of the most readily available ECM protein, serum fibronectin, has been found to reside in the RGD sequence. Subsequently, it was shown that a number of ECM proteins contain the RGD motif, and that the sequence is required for recognition and interaction of these proteins with the cell. (Ruoslahti et al., Science, 238:491, 1987.)
Further research revealed that immobilized synthetic peptides containing the RGD motif can mimic the cell attachment activity of ECMs. In addition, in solution these same peptides were capable of inhibiting cell attachment to other RGD-containing ligands. Thus, RGD-based therapeutics can potentially function either as agonists which promote the interaction of cells and tissues with artificial matrices containing the RGD-based drug, or as antagonists which inhibit cell-cell and cell-ECM interactions.
Abnormal ECM function has been associated with a number of diseases or conditions, such as cardiovascular disease, cancer, osteoporosis, and inflammation. For example, RGD plays an integral role in the formation of blood clots which can give rise to cardiovascular diseases.
Initial events in blood-clot formation, i.e., thrombus formation, frequently entail the activation of platelets by thrombogenic surfaces containing RGD sequences, and the subsequent aggregation of platelets onto these surfaces. A protein complex on the surface of platelet cells has been shown to interact with the RGD motif. Adhesion and aggregation of platelets is mediated by adhesive proteins that interact with the platelet membrane glycoprotein complex .alpha..pi.b.beta.3 at the platelet surface. Platelet .alpha..pi.b.beta.3 glycoprotein complex is a member of the family of cell adhesion receptors, called integrins. (Ruoslahti, E. J. Clin. Invest., 87:1, 1991.) It has been shown that, like several of the integrins, the .alpha..pi..beta.3 complex on activated platelets can bind to a RGD tripeptide sequence in several proteins including fibrinogen, fibronectin, von Willebrand factor, and vitronectin. Molecules containing the RGD motif or analogs of the RGD motif can inhibit the binding of fibrinogen to the .alpha..pi.b.beta.3 complex on activated platelets.
The antithrombotic activity of many molecules that inhibit the .alpha..pi.b.beta.3-fibrinogen interaction has been accessed in vitro and in vivo. Most of these compounds fall into four categories: RGD-based peptides (small linear and cyclic peptides containing the RGD sequence or its equivalent), (Cheng et al., J. Med. Chem. 37:1, 1994) snake venom peptides, monoclonal antibodies raised against .alpha..pi.b.beta.3, and non-peptide fibrinogen receptor antagonists that mimic the RGD tripeptide sequence. (Weller et al., Drugs of the Future 19:461, 1994.)
The second peptide motif, ELR, is found in chemokines. Chemokines are small proteins having important roles in a wide range of acute and chronic inflammatory processes. The C-X-C chemokine family is characterized by a four cysteine motif, in which the first two cysteines are separated by a single intervening residue, the other two appearing elsewhere in the protein sequence (Horuk, R., TIPS 15:159 (1994).) In general, C-X-C chemokines are chemoattractants for neutrophils--white blood cells which are involved in inflammatory responses. Hence, C-X-C chemokines are considered to be the key mediators of inflammatory responses where neutrophil recruitment is involved. Interleukin 8, Il-8, is a potent inflammatory mediator that belongs to the C-X-C family which also includes MGSA (melanoma growth-stimulating activity) and NAP-2 (neutrophil activating peptide).
A specific tripeptide motif, ELR, occurs close to the N-terminus in all C-X-C chemokines that demonstrate biological activation of neutrophils. Although not the sole determinant for binding of the C-X-C chemokines to cellular receptors, the ELR motif is essential for the binding of the C-X-C chemokines to cellular receptor. (Moser, B. et al., J. Biol. Chem., 268:7125 (1993); Rajarathnam et al., Biochem., 33:6623 (1994) and Clark, Lewis I. et al., J. Biol. Chem. 266:23128 (1991).) Molecules which compete for the ELR binding site to the C-X-C chemokine receptor should antagonize the binding of the endogenous ligand and hence prove to be useful in the treatment of C-X-C chemokine driven diseases.
Thiazolidinones are another class of compounds of biological interest. They have been reported to possess a wide range of biological activities including antifungal, antibacterial, antihistaminic, antimicrobial, anti-inflammatory and antidiabetic activities. (Singh, S. P.; Parmar, S. S.; Raman, K.; Stenberg, V. I., Chem. Rev. 1981, 81, 175-203 and Berger, J.; Biswas, C.; Hayes, N.; Ventre, J.; Wu, M.; and Doebber, T. W., Endocrinology, 1995, 137, 1984.)
None of the references described herein is admitted to be prior art.